PROJECT SUMMARY The overall goal of this proposal is to equip the Advanced Light Microscopy/Spectroscopy (ALMS) core facility of the UCLA California NanoSystems Institute (CNSI) with the next generation of multiphoton microscope (MPM) for imaging physiological processes in vivo and for deep imaging of CLARITY-treated tissues. The intent is to replace two older MP microscopes that have reached the end of their useful life and are no longer serviceable/upgradable. The new MPM will complement other fluorescence imaging technologies, such as wield- field microscopy, confocal microscopy and light-sheet microscopy, which are already available within the ALMS Core. Many important new experimental avenues will be made possible by utilizing the new MPM on an upright geometry platform equipped for complex cellular manipulation using electrophysiology and microinjection. MP microscopy is a powerful technique based on nonlinear interactions between photons and matter. As such, MP microscopy provides distinct advantages over other fluorescence microscopy approaches. MP microscopy allows 3D optical sectioning capability without the need for a confocal pinhole and can image effectively 6-fold deeper than confocal microscopy using the same sample and fluorophores. This improvement in imaging depth arises because MP excitation with infrared light is much less sensitive to scattering of the excitation and emission photons. It also has the advantage of reduced out-of-focus photobleaching and reduced phototoxicity. This makes MP microscopy ideal for imaging structures in vivo in a living animal and deep into thick scattering specimen. The research projects described in this proposal come from a wide spectrum of UCLA Faculty with different research interests. They all take advantage of the MPM's exceptional ability to image cells at high resolution at greater depths in live and fixed tissues. This is particularly useful for investigators who are interested in studying phenomenon at single-cell resolution while maintaining the structural integrity of large tissues and organs. Several of the major users propose to study the neuro-anatomical complexity of the brain (Drs. Evans and Walwyn), the spinal cord (Drs. MacKenzie-Graham and Edgerton), the heart (Drs. Ajijola and Tompkins) or the colon (Dr. Tache) by tracing neural projections in various human disease models. Others will study dynamic interactions of immune cells (Drs. Belperio and Butte) or cancer cells (Dr. Bentolila) in vivo in small rodent models. Two users (Drs. Di Carlo and Li) propose to study the neo-vascularization of biomaterial and one (Dr. Xia) the biodistribution and toxicity of nanoparticles in vivo. The availability of the MP microscope will significantly enhance and expand the research directions of these investigators, most of whom have ongoing NIH-funded research programs, and will greatly add to their ability to study physiological properties of individual cells within the more complex framework of intact live and fixed tissues. The requested MP microscope will be the ONLY instrument available on a shared-user basis for the entire UCLA campus.